Photosynthesis is an important component of upland cotton (Gossypium hirsutum L.) yield, but little has been done to increase the photosynthetic performance within the cotton germplasm pool. Part of this dilemma is due to the multi-component aspect of this process and also to lack of information on genetic variation among such components. The objectives of this research were to identify genetic variability in photosynthetic components for six cotton genotypes previously shown to differ in leaf CO2-exchange rates (CER) and to determine if an afternoon decline in photosynthesis altered genotypic differences in CER. CO2-exchange rates were measured at several internal CO2 levels (Ci) to generate CER vs. Ci curves for each genotype and thereby isolate some of the components of photosynthesis. Ribulose 1,5 bisphosphate carboxylase-oxygenase (Rubisco), hydroxypyruvate reductase, malate dehydrogenase, and catalase activities were assayed on leaves used to generate the CER vs. Ci curves. Ambient CER and chlorophyll (Chl) fluorescence measurements were taken before and after solar noon to test for an afternoon decline in photosynthesis. Dixie King, a low ambient CER genotype, exhibited a greater CO2 compensation point, lower carboxylation efficiency, and reduced Photosystem II (PS II) activity than the other genotypes. The carboxylation efficiency of DES 119 was 13% greater than STV 508 and 29% greater than Dixie King, but not different from the other genotypes in 1994. Pee Dee 3 had greater maximum assimilation rate (A) than all other genotypes except STV 213 in 1993. Although no significant genotype by time of day interaction was detected, CER and Chl fluorescence variable to maximum ratio (Fv/Fm) were reduced 8% and 39%, respectively, in the afternoon as compared to the morning. This study demonstrates genetic variations in many of the components of photosynthesis. However, the narrow range of variation in such components for superior photosynthesizing genotypes explains why difficulties are encountered when breeding for increased photosynthesis.